TW202210090A - Pharmaceutical compositions and uses thereof in treating muscle atrophy - Google Patents

Abstract

Disclosed herein are pharmaceutical compositions and uses thereof in treating muscle atrophy. According to some embodiments of the present disclosure, the pharmaceutical composition comprises an effective amount of ethanol extracts of Artemisia argyi, Morus alba L., Leonurus japonicus Houtt, Capsicum annuum L., Lophatherum gracile Brongn, Curcuma longa, and Glycyrrhiza uralensis; and a pharmaceutically acceptable excipient. According to certain embodiments of the present disclosure, the ethanol extracts comprise 14 ingredients, including chlorogenic acid, leonurine, schaftoside, rutin, isoschaftoside, isochlorogenic acid, 4,5-dicaffeoylquinic acid, quercetin, apigenin, glycyrrhizic acid, bisdemethoxycurcumin, demethoxycurcumin, curcumin, and artemisetin.

Description

Pharmaceutical composition and use thereof in the treatment of sarcopenia

The present disclosure is in the field of treating disease. More specifically, the present disclosure relates to novel pharmaceutical compositions and their use in the treatment of muscle atrophy.

Adequate skeletal muscle plasticity is important for individual activity, and insufficient muscle function can seriously affect individual quality of life, morbidity, and mortality. Sarcopenia, also known as muscular dystrophy, is a disease characterized by loss of muscle tissue and progressive muscle weakness. The occurrence of sarcopenia is related to different factors such as long-term inactivity, genetic inheritance, aging, malnutrition, drugs and/or various injuries or diseases affecting the musculoskeletal or nervous system. The symptoms of sarcopenia vary depending on the cause and the severity of muscle loss. In addition to loss of muscle mass, symptoms of sarcopenia include difficulty or loss of physical activity (such as standing, walking, climbing or balance), curvature of the spine (scoliosis), heart problems, swallowing problems, and even breathing problems.

Physical therapy (including specific stretches and exercises) may provide some benefit to people with sarcopenia by increasing muscle strength, improving blood circulation, reducing spasms, and preventing immobility. Functional electrical stimulation (FES) is an alternative therapy for sarcopenia. FES primarily uses electrical impulses to stimulate the contraction of muscles in the affected area. However, these treatments only slow the progression of muscle loss, not reverse the recovery or cure sarcopenia. In the treatment of patients with severe sarcopenia, anabolic steroids (or anabolic-androgenic steroids (AAS; such as hydroxymethylandrogenone) can be administered by increasing the protein synthesis, sugar production, and muscle strength to alleviate the symptoms associated with sarcopenia. However, these anabolic steroids tend to have varying degrees of side effects that limit their clinical use, such as kidney problems or kidney failure, liver damage (e.g. , liver cirrhosis), heart problems (eg, enlarged heart, high blood pressure, changes in blood cholesterol, arterial damage, stroke, and heart disease), blood clots, cancer (eg, testicular or prostate cancer), infections (eg, hepatitis or human immunity) Defective viral infection), hormonal changes, and psychiatric problems (eg, aggression, mania, and delusions).

In view of this, a novel method and/or agent for treating sarcopenia is urgently needed in the related art.

SUMMARY The purpose of this summary is to provide a simplified summary of the disclosure to give the reader a basic understanding of the disclosure. This summary is not an exhaustive overview of the disclosure, and it is not intended to identify key/critical elements of embodiments of the invention or to delineate the scope of the invention.

The present disclosure relates to novel pharmaceutical compositions and their use in the treatment of sarcopenia.

A first aspect of the present disclosure relates to a pharmaceutical composition comprising an ethanolic extract of a Chinese herbal medicine mixture, and a pharmaceutically acceptable excipient; wherein the Chinese herbal medicine mixture is composed of Artemisia argyi , mulberry ( Morus alba L. ), motherwort ( Leonurus japonicus Houtt ), bell pepper ( Capsicum annuum L. ), pale bamboo leaves ( Lophatherum gracile Brongn ), turmeric ( Curcuma longa ) and licorice ( Glycyrrhiza uralensis ).

According to certain embodiments of the present disclosure, the ethanol extract is at a temperature of about 30-100° C., using ethanol to extract 0.5-5% of mugwort, mulberry, motherwort, bell pepper, pale bamboo leaves, sichuan turmeric and licorice. prepared in hours.

According to some preferred embodiments, the ethanol extract is at a temperature of about 50-80 ° C, using 95% ethanol to mugwort leaves, mulberry leaves, motherwort leaves, bell pepper leaves, pale bamboo leaves. It is prepared by extracting the leaves, turmeric root and licorice root for 3-5 hours.

According to certain embodiments, during the extraction process, the leaves of mugwort are mixed in a weight ratio of about 4-6:4-6:4-6:2-3:2-3:1:1 , mulberry leaves, motherwort leaves, bell pepper leaves, pale bamboo leaves, sichuan turmeric roots and licorice roots. According to a specific embodiment, the leaves of mugwort, the leaves of mulberry, the leaves of motherwort, the leaves of bell pepper, the leaves of pale bamboo, the leaves of Sichuan Turmeric root and licorice root.

According to certain embodiments, the prepared ethanol extract contains 14 kinds of Chinese herbal medicine ingredients, including chlorogenic acid, leonurine, schaftoside, rutin, isoschaf schaftoside, chlorogenic acid, 4,5-dicaffeoylquinic acid, quercetin, apigenin, glycyrrhizin ( glycyrrhizic acid), bisdemethoxycurcumin, demethoxycurcumin, curcumin and artemisetin. According to certain embodiments, the ethanolic extract containing Chinese herbal medicine can treat sarcopenia by modulating the expression of different molecules in the pathogenic pathway of sarcopenia and/or improving mitochondrial function.

A second aspect of the present disclosure therefore relates to a pharmaceutical composition comprising monochlorogenic acid, leonurine, savutoside, rutin, isoxafutoside, isochlorogenic acid, 4,5-dicaffeoside quinic acid, quercetin, apigenin, glycyrrhizin, didemethoxycurcumin, demethoxycurcumin, a mixture of curcumin and einstein; and a pharmaceutically acceptable excipient.

According to certain embodiments of the present disclosure, the mixture comprises 5-10 weight percent (wt%) chlorogenic acid, 0.1-2 weight percent leonurine, 0.1-2 weight percent saftoside, 5-10 wt% rutin, 35-45 wt% isoxafutoside, 20-30 wt% isochlorogenic acid, 3-6 wt% 4,5-dicaffeoquinic acid, 0.1-0.5 wt% Quercetin, 1-3 weight percent apigenin, 1-3 weight percent glycyrrhizin, 1-3 weight percent didemethoxycurcumin, 1-3 weight percent demethoxycurcumin , 5-10 weight percent of curcumin, and 0.1-0.5 weight percent of eflavin.

In some preferred embodiments, the mixture comprises 7-8 weight percent chlorogenic acid, 0.5-1 weight percent Leonurine, 0.5-1.5 weight percent savutoside, 7-8 weight percent ruta glycosides, 38-42 weight percent isoxafutoside, 20-25 weight percent isochlorogenic acid, 4-5 weight percent 4,5-dicaffeoquinic acid, 0.1-0.3 weight percent quercetin , 1-2 weight percent apigenin, 1-2 weight percent glycyrrhizin, 2-3 weight percent two-demethoxy curcumin, 2-3 weight percent demethoxycurcumin, 5-7 Curcumin in weight percent, and 0.1-0.3 weight percent eflavin.

The present disclosure also provides methods of treating sarcopenia in a subject utilizing the pharmaceutical compositions of the present invention. The method comprises administering to the individual an effective amount of a pharmaceutical composition of the present invention.

According to certain embodiments of the present disclosure, the pharmaceutical composition is administered orally or topically to the individual. In a preferred embodiment, the pharmaceutical composition is administered to the individual daily for at least 7 days; more preferably, for at least 14 days. In a specific embodiment, the pharmaceutical composition is administered to the individual daily for 14 days.

A subject suitable for treatment with the pharmaceutical compositions and/or methods of the present invention is a mammal; preferably a human.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit and other purposes of the present invention, as well as the technical means and implementation aspects of the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description for the embodiments and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The features of various specific embodiments as well as method steps and sequences for constructing and operating these specific embodiments are encompassed in the detailed description. However, other embodiments may also be utilized to achieve the same or equivalent function and sequence of steps.

i. definition

Notwithstanding that the numerical ranges and parameters setting forth the broader scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains the standard deviation resulting from individual testing methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value lies within an acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. Except for the experimental examples, or unless expressly stated otherwise, all ranges, quantities, values and percentages used herein should be understood when used to describe material amounts, time periods, temperatures, operating conditions, quantity ratios and other similar ) are modified by "Covenant". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying claims are approximate numerical values and may be changed as required. At a minimum, these numerical parameters should be construed to mean the number of significant digits indicated and the numerical values obtained by applying ordinary rounding. Numerical ranges are expressed herein as being from one endpoint to the other or between the endpoints; unless otherwise indicated, the numerical ranges recited herein are inclusive of the endpoints.

Unless otherwise defined in this specification, scientific and technical terms used herein have the same meanings as understood and commonly used by those of ordinary skill in the art to which this invention belongs. In addition, unless contradicting the context, the singular noun used in this specification covers the plural form of the noun; and the plural noun used also covers the singular form of the noun.

The term "atrophy" in the present disclosure refers to a reduction in the size and/or number of cells of a tissue (eg, a muscle tissue) or organ after reaching normal size. "Muscle atrophy" or "muscle atrophy" in the present disclosure refers to a decrease in muscle mass and strength. Sarcopenia can be the loss of some or all of the muscle and can occur in any part of an individual's muscles, including skeletal muscles (eg , biceps, quadriceps, gastrocnemius, tibialis anterior, forearm, abdominal, and paraspinal muscles) muscles) and non-skeletal muscles (eg, diaphragm and extraocular muscles).

In this disclosure, the term "extract" includes both crude extract and refined extract. Specifically, the crude extract is the product obtained from a simple extraction by contacting selected plant parts with at least one extractant (ie, extraction solvent). In optional cases, the resulting crude extract may subsequently be subjected to one or more separation and/or purification treatments to obtain a refined extract. Plant extracts can be in liquid form (eg, solutions, concentrates, distillates) or solids (eg, pastes, granules, or powders) from which the solvent has been removed.

The term "weight percentage" (wt %) in the present disclosure refers to a mixture comprising an ingredient (eg, chlorogenic acid, leonurine, savutoside, Rutin, Isosafutoside, Isochlorogenic Acid, 4,5-Dicaffeic Acid, Quercetin, Apigenin, Glycyrrhizin, Didemethoxycurcumin, Demethoxycurcumin, weight percent of curcumin or eflavin). Weight percent (wt %) is obtained by dividing the weight of the ingredient by the total weight of the mixture and is expressed as a percentage and/or a decimal.

In this disclosure, the term "weight ratio" refers to each component (eg, leaves of mugwort, leaves of mulberry, leaves of motherwort, leaves of bell pepper, leaves of pale bamboo, leaves of turmeric root and licorice root) in a mixture (eg, the Chinese herbal medicine mixture of the present disclosure) in individual amounts, and expressed as the weight ratio of each component.

In this disclosure, the term "treat" includes partial or complete prevention, amelioration, alleviation and/or management of symptoms, secondary diseases or symptoms associated with sarcopenia. The term "treat" in this specification also refers to the application or administration of a pharmaceutical composition of the present disclosure to a subject suffering from a symptom, secondary disease or condition associated with sarcopenia to achieve partial Or completely alleviate, slow, cure the disease, delay the onset, inhibit the progression of the disease, reduce the severity of the disease, and/or reduce the occurrence of one or more symptoms, secondary diseases or symptoms associated with sarcopenia. Symptoms, secondary diseases or conditions associated with sarcopenia include, but are not limited to, decreased muscle mass and/or strength, difficulty or loss of physical activity (such as standing, walking, climbing or balance), spinal curvature (spine scoliosis), heart problems, swallowing problems, and breathing problems. "Treatment" can also be administered to an individual with an early stage of such signs or symptoms to reduce the occurrence of symptoms, secondary diseases or symptoms associated with sarcopenia. A treatment is "effective" when one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" when the progression of a symptom, secondary disease or symptom is slowed or stopped.

"Effective amount" as used herein refers to an amount of an agent sufficient to produce the desired therapeutic response. An effective amount also refers to a compound or composition whose toxic or detrimental effects are outweighed by its therapeutically beneficial effects. An effective amount of an agent does not necessarily cure the disease or condition, but can delay, retard or prevent the occurrence of the disease or condition, or alleviate the symptoms associated with the disease or condition. A therapeutically effective amount can be divided into one, two or more doses and administered one, two or more times over a specified period in the appropriate dosage form. The specific therapeutically effective amount depends on a variety of factors, such as the particular condition to be treated, the physiological condition of the individual (eg, the individual's weight, age, or gender), the type of individual being treated, the duration of treatment, and concurrent treatments (if any) nature and the specific formulation used and the structure of the compound or its derivatives. For example, a therapeutically effective amount can be expressed as the total weight of the active ingredient, such as in grams, milligrams or micrograms; or as a ratio of the weight of the active ingredient to body weight, such as in milligrams per kilogram of body weight (mg/kg body weight). kg). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (eg, a mixture of specific compounds of the pharmaceutical composition of the present invention, or an ethanolic extract of a mixture of Chinese herbal medicines), such as molar, weight, volume, weight mol Ear concentration, molar fraction, weight fraction and mixing ratio. Those skilled in the art can calculate the human equivalent dose (HED) of an agent (eg, a pharmaceutical composition of the present disclosure) based on the dose in the animal model. For example, those skilled in the art can use the "Estimating the Maximum Safe Initial Dose for Adult Healthy Volunteers in Initial Clinical Treatment Tests" published by the US Food and Drug Administration (FDA). Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the highest safe dose for human use.

"Subject" and "patient" are used interchangeably in this disclosure, and refer to animals, including humans, that can be treated with the pharmaceutical compositions and/or methods of the present invention. Unless otherwise specified, the term "subject" or "patient" means both male and female. Accordingly, the term "subject" or "patient" includes any mammal that can produce a therapeutic benefit after receiving a pharmaceutical composition or method of treatment of the present disclosure. Exemplary "subject" or "patient" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds and poultry . In an exemplary embodiment, the individual is a human.

II. Detailed Description of the Invention

(i) pharmaceutical composition

The present invention aims to provide a method for treating sarcopenia in a subject, and a pharmaceutical formulation or dietary supplement for carrying out the method.

Accordingly, a first aspect of the present disclosure is directed to a pharmaceutical composition that can be used to treat an individual suffering from or suspected of suffering from sarcopenia. The pharmaceutical composition comprises an ethanol extract of a Chinese herbal medicine mixture, and a pharmaceutically acceptable excipient; wherein the Chinese herbal medicine mixture is composed of Artemisia argyi (also known as silver wormwood) or Chinese wormwood (Artemisia argyi). Chinese mugwort), mulberry ( Morus alba L. , also known as white mulberry), motherwort ( Leonurus japonicus Houtt , also known as Chinese motherwort), pepper ( Capsicum annuum L. , also known as motherwort) It is chili or pepper), Lophatherum gracile Brongn , Curcuma longa (also known as turmeric) and Glycyrrhiza uralensis (also known as Chinese liquorice) ) is composed of.

According to certain embodiments, at a suitable temperature, mixing mugwort, mulberry, motherwort, bell pepper, pale bamboo leaves, sichuan turmeric and licorice, and extracting with a solvent (for example, water or ethanol) for a period of time, to prepare the composition of the present invention. Extracts of active ingredients (that is, those with therapeutic benefits for sarcopenia).

Depending on the desired purpose, the solvent used to extract the Chinese herbal medicine (ie, the combination of mugwort, mulberry, motherwort, bell pepper, pale bamboo leaf, sichuan turmeric and licorice) may be a supercritical fluid (SFC); such as carbon dioxide , water, methane, ethane, propane, ethylene, propylene, methanol, ethanol and acetone), water, C _1-4 alcohols (such as ethanol, 1-propanol, n-butanol, isobutanol and tert-butanol) , acetone, ethyl acetate, n-hexane, or a combination thereof. According to certain embodiments, at a temperature of 30-100°C (eg 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100°C) , at 75-100% (eg 96, 97, 98, 99 or 100%) ethanol extraction of the herbal medicine for 0.5-5 hours (eg 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 hours) ) to obtain the extract of the pharmaceutical composition of the present invention.

According to certain embodiments, after mixing mugwort leaves, mulberry leaves, motherwort leaves, bell pepper leaves, pale bamboo leaves, sichuan turmeric roots and licorice roots, at a temperature of about 50-80 ° C. Next, the mixed Chinese herbal medicine components or the Chinese herbal medicine mixture are extracted with 95% ethanol for about 3-5 hours to obtain the extract of the pharmaceutical composition of the present invention. In an exemplary embodiment, the herbal mixture or herbal components (i.e., leaves of mugwort, leaves of mulberry, leaves of motherwort, leaves of bell pepper are treated with 95% ethanol at a temperature of about 70° C. leaves, the leaves of the pale bamboo leaves, the roots of sichuan turmeric and the roots of licorice) are extracted for about 4 hours to obtain the extract of the pharmaceutical composition of the present invention.

According to certain embodiments of the present disclosure, the Chinese herbal medicine component is about 4-6 (eg, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6): 4-6 (eg, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3 , 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6): 4-6 (for example, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6): 2-3 (eg, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3): 2-3 (eg, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3): 1:1 weight ratio mixing. As can be imagined, those skilled in the art can adjust the proportions of the Chinese herbal medicine components according to the desired purpose. For example, the herbal components may be mixed in a weight ratio of about 4:4:4:2:2:1:1. Alternatively, the herbal components may be mixed in a weight ratio of about 4.5:5:6:2.5:2:1:1. Alternatively, the Chinese herbal medicine components may be mixed in a weight ratio of about 5:5:5:2:3:1:1. According to some embodiments of the present disclosure, the Chinese herbal medicine components are mixed and extracted in a weight ratio of about 5:5:5:2.5:2.5:1:1 to obtain a crude extract. The crude extract can then be filtered, concentrated and/or freeze-dried to obtain a crude extract powder or paste. Alternatively, it can be further purified (eg, tube injection chromatography or precipitation) to obtain a refined extract.

According to certain embodiments, the resulting extract contains active ingredients that can be used to treat sarcopenia; the active ingredients include at least chlorogenic acid, leonurine, saftoside, rutin, isosafutoside, isogreen Orthoic acid, 4,5-dicaffeoquinic acid, quercetin, apigenin, glycyrrhizin, didemethoxycurcumin, demethoxycurcumin, curcumin, and eflavin.

的結構； (2) 益母草鹼，具有

的結構； (3) 沙夫托甙，具有

的結構； (4) 芸香苷，具有

的結構； (5) 異沙夫托甙，具有

的結構； (6) 異綠原酸，具有

的結構； (7) 4,5-二咖啡奎寧酸，具有

的結構； (8) 槲皮素，具有

的結構； (9) 芹菜素，具有

的結構； (10) 甘草酸苷，具有

的結構； (11) 二去甲氧基薑黃素，具有

的結構； (12) 去甲氧基薑黃素，具有

的結構； (13) 薑黃素，具有

的結構；以及 (14) 艾黃素，具有

的結構。Accordingly, a second aspect of the present disclosure provides a pharmaceutical composition comprising a mixture of a plurality of compounds, and a pharmaceutically acceptable excipient, wherein the mixture is composed of 14 active ingredients, comprising: (1) Chlorogenic acid, with

The structure of ; (2) Leonurine, with

The structure of ; (3) Saftoside, with

The structure of ; (4) Rutin, with

The structure of ; (5) Isosafutoside, with

The structure of ; (6) isochlorogenic acid, with

The structure of ; (7) 4,5-dicaffeoquinic acid, with

The structure of ; (8) Quercetin, with

The structure of; (9) Apigenin, with

The structure of ; (10) Glycyrrhizin, with

The structure of; (11) Didemethoxycurcumin, with

The structure of ; (12) Demethoxycurcumin, with

The structure of; (13) Curcumin, with

The structure of ; and (14) eflavin, with

Structure.

According to some embodiments, (1) In the mixture, the weight of chlorogenic acid is about 5-10% (eg, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% by weight of the total weight of the mixture) ), namely 5-10 wt%; (2) In the mixture, the weight of Leonurine is about 0.1-2% of the total weight of the mixture (for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2%), i.e., 0.1-2 wt%; (3) In the mixture, the weight of saftoside is about 0.1-2% of the total weight of the mixture (for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2%), i.e. 0.1-2 wt%; (4) In the mixture, the weight of rutin is about 5-10% of the total weight of the mixture (eg, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10%) , ie 5-10 wt%; (5) In the mixture, the weight of isoxafutoside is about 35-45% of the total weight of the mixture (for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5 or 45%), i.e. 35-45 wt%; (6) In the mixture, the weight of isochlorogenic acid accounts for about 20-30% of the total weight of the mixture (for example, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 , 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5 or 30%), i.e. 20-30 wt%; (7) In the mixture, the weight of 4,5-dicaffeoquinic acid is about 3-6% (eg, 3, 3.5, 4, 4.5, 5, 5.5 or 6%) by weight of the total weight of the mixture, i.e. 3-6 wt%; (8) In the mixture, the weight of quercetin is about 0.1-0.5% (eg, 0.1, 0.2, 0.3, 0.4 or 0.5%) of the total weight of the mixture, i.e. 0.1-0.5 wt%; (9) In the mixture, the individual weights of apigenin, glycyrrhizin, didemethoxycurcumin and demethoxycurcumin are respectively about 1-3% of the total weight of the mixture (for example, 1, 2 or 3%), i.e. 1-3 wt%; (10) In the mixture, the weight of curcumin is about 5-10% (eg, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10%) of the total weight of the mixture , i.e. 5-10 wt%; and (11) In the mixture, the weight of eflavin is about 0.1-0.5% (eg, 0.1, 0.2, 0.3, 0.4, or 0.5%) of the total weight of the mixture, ie, 0.1-0.5 wt%.

According to certain exemplary embodiments, the mixture comprises 7-8 weight percent chlorogenic acid, 0.5-1 weight percent Leonurine, 0.5-1.5 weight percent savutoside, 7-8 weight percent rutin, 38-42 weight percent of isosafutoside, 20-25 weight percent of isochlorogenic acid, 4-5 weight percent of 4,5-dicaffeoquinic acid, 0.1-0.3 weight percent of quercetin, 1 -2 weight percent apigenin, 1-2 weight percent glycyrrhizin, 2-3 weight percent didemethoxycurcumin, 2-3 weight percent demethoxycurcumin, 5-7 weight percent of curcumin, and 0.1-0.3 weight percent of eflavin.

According to a specific embodiment, the mixture comprises 7.7 weight percent chlorogenic acid, 0.8 weight percent Leonurine, 1 weight percent savutoside, 7.2 weight percent rutin, 40.9 weight percent isosafutoside, 23.8 weight percent isochlorogenic acid, 4.8 weight percent 4,5-dicaffeoquinic acid, 0.2 weight percent quercetin, 1.2 weight percent apigenin, 1.8 weight percent glycyrrhizin, 2.2 weight percent Didemethoxycurcumin, 2.2 wt% demethoxycurcumin, 6 wt% curcumin, and 0.2 wt% eosin.

The pharmaceutical composition of the present disclosure is preferably freeze-dried and stored in a low temperature and dry environment for later use.

Depending on the purpose of implementation, the pharmaceutical composition of the present disclosure can be formulated with one or more suitable pharmaceutically acceptable carriers or excipients, and the pharmaceutical composition of the present disclosure can be formulated as a solid, semi-solid or liquid dosage form Forms such as pills, tablets, capsules, powders, pastes, granules and ointments. As such, the active ingredient (eg, the herb or an extract of the herb component, or a mixture of the compounds) can be administered orally, bucally, topically, and parenterally. In pharmaceutical dosage forms, the pharmaceutical compositions of the present disclosure can be administered to a subject alone or in combination with other pharmaceutically active agents known to treat sarcopenia. Different dosage forms suitable for each route of administration are well known to those skilled in the art. It should be noted that the optimal route of administration will vary with the type and severity of the disease or condition.

In certain embodiments, the pharmaceutical compositions of the present disclosure are in dosage forms suitable for non-oral administration, such as topical administration or injection, including, but not limited to, subcutaneous, intramuscular, intraperitoneal, and intravenous injection. Pharmaceutical compositions can be formulated as oily or aqueous isotonic suspensions, solutions or emulsions, and can contain formulatoary agents such as suspending, stabilizing or dispersing agents. Alternatively, the compositions may be prepared in dry form, such as powder, crystals, or freeze-dried solids, for administration with sterile, pyrogen-free water or isotonic saline prior to use. The compositions may also be presented in sterile ampoules or vials.

In certain embodiments, the pharmaceutical compositions of the present disclosure are administered orally in solid dosage form. These solid dosage forms can be capsules, pouches, lozenges, pills, lozenges, powders or granules. In these dosage forms, the pharmaceutical composition of the present invention (comprising chlorogenic acid, leonurine, safutoside, rutin, isosafutoside, isochlorogenic acid, 4,5-dicaffeoquinic acid, quercetin, apigenin, glycyrrhizin, didemethoxycurcumin, demethoxycurcumin, curcumin, and eflavin) in admixture with at least one pharmaceutically acceptable excipient. Any of the above solid dosage forms may optionally contain coatings and shell layers, such as enteric coatings and coatings to improve the release rate of any ingredient. Examples of such coatings are well known in the art. In one embodiment, the pharmaceutical composition of the present disclosure is a tablet, such as a rapid release tablet. In another embodiment, the pharmaceutical composition of the present disclosure is formulated as a prolonged release dosage form. In yet another embodiment, the pharmaceutical compositions of the present disclosure are powders encapsulated in soft and hard gelatin capsules.

In certain embodiments, the pharmaceutical compositions of the present disclosure are administered orally in liquid dosage form. Liquid dosage forms may further comprise a buffering agent to maintain a desired pH. Liquid dosage forms can also be filled in soft gelatin capsules. For example, the liquid may comprise a solution, suspension, latex, microslip, sediment or any desired liquid medium which carries the active ingredients of the pharmaceutical composition. Liquids can be designed to modify the active ingredients of the pharmaceutical compositions of the present invention to form a drug-containing latex or dispersed dispersion. According to one embodiment of the present disclosure, the active ingredient of the pharmaceutical composition of the present invention is formulated in a solvent comprising 95% olive oil and 5% glycerol for oral administration.

(ii) Uses of the pharmaceutical compositions of the present disclosure

A second aspect of the present disclosure relates to a method for treating sarcopenia in a subject. The method comprises administering to the individual an effective amount of a pharmaceutical composition of the present disclosure.

In certain embodiments, a pharmaceutical composition of the present disclosure is administered topically to the individual. Alternatively, a pharmaceutical composition of the present disclosure is administered orally to the individual. However, the present disclosure is not limited thereto.

According to embodiments of the present disclosure, one or more doses (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) are administered orally or topically to the individual or more doses) of the pharmaceutical composition of the present invention.

In one embodiment, the individual is a mouse.

In order to produce the therapeutic effect, each dose of the pharmaceutical composition of the present invention is about 1-1,000 mg/kg body weight; preferably, the pharmaceutical composition of the present invention is about 10-100 mg/kg body weight per dose; More preferably, administration of about 50-70 mg/kg body weight of the pharmaceutical composition of the present invention per dose is sufficient to produce a therapeutic effect in an individual suffering from sarcopenia. In a specific embodiment, the pharmaceutical composition of the present invention is administered to an individual at 60 mg/kg body weight per dose to produce a therapeutic effect.

Those skilled in the art can determine the human equivalent dose of the pharmaceutical composition of the present invention based on the administered dose of the animal experiments provided in the embodiments of the present disclosure. Accordingly, the effective dosage of the pharmaceutical composition of the present invention for human subjects is between 0.1-100 mg/kg body weight per dose; preferably, 1-10 mg/kg body weight per dose. In a preferred embodiment, the human equivalent dose is about 4-6 mg per kilogram of body weight per dose.

According to certain preferred embodiments, the pharmaceutical composition of the present disclosure is administered to the individual daily for at least 7 days; for example, the pharmaceutical composition of the present disclosure is administered to the individual daily for a total of 7, 8, 9, 10, 11, 12, 13, 14 or more days. In a specific embodiment, the pharmaceutical composition of the present disclosure is administered to the subject daily for 14 days.

As is conceivable, the skilled person or clinical operator may adjust the dosage and duration of treatment according to the patient's physiology or the severity of the disease.

Depending on the desired purpose, the pharmaceutical compositions of the present invention may be administered to a subject alone or in combination with other therapeutically beneficial treatments for sarcopenia. The pharmaceutical compositions of the present invention may be administered to an individual before, concurrently with, or after administration of the other treatment.

A subject that can be treated with the pharmaceutical compositions and/or methods of the present invention is a mammal; for example, humans, mice, rats, tenrens, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats , cows and rabbits. Preferably, the individual is a human.

Several experimental examples are provided below to illustrate certain aspects of the present invention, so as to facilitate the practice of the present invention by those skilled in the art to which the present invention pertains, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art, after reading the description presented herein, can fully utilize and practice the present invention without undue interpretation. All publications cited herein are considered part of this specification in their entirety. Example

Materials and Methods

Preparation of the pharmaceutical composition of the present invention (ATG-125)

At room temperature, seven kinds of Chinese herbal ingredients, including mugwort leaves (512 grams), mulberry leaves (512 grams), motherwort leaves (512 grams), bell pepper leaves (256 grams), bamboo leaves leaves (256 g), turmeric root (103 g) and licorice root (103 g), soaked in 15 liters of 95% ethanol for one day, and then filtered. The filtrate was retained, and the herbal ingredients were soaked again in the same way. After the filtrate was combined, it was placed in a 70°C water bath for extraction for 4 hours, and filtered again. Repeat the extraction step two more times. All extracts were combined and concentrated under reduced pressure. The following yield was obtained: 331.11 g (14.69%).

According to the analysis results of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS), the obtained ethanol extract contains chlorogenic acid (weight % ethanol) 0.38% of the total weight of the extract), Leonurine (0.04% of the total weight of the ethanol extract), Shaftuoside (0.05% of the total weight of the ethanol extract), Rutin (weight of the total weight of the ethanol extract) 0.36% by weight), isoxafutoside (2.03% by weight based on the total weight of the ethanol extract), isochlorogenic acid (1.18% by weight based on the total weight of the ethanol extract), 4,5-dicaffeoquinic acid (0.24% by weight of the total weight of the ethanol extract), quercetin (0.01% by weight of the total weight of the ethanol extract), apigenin (0.06% by weight of the total weight of the ethanol extract), glycyrrhizin (by weight of the total weight of the ethanol extract) 0.09% of the total weight of the ethanol extract), Didemethoxycurcumin (0.11% of the total weight of the ethanol extract), Demethoxycurcumin (0.11% of the total weight of the ethanol extract), Turmeric Ephedrine (0.3% by weight based on the total weight of the ethanolic extract) and eflavin (0.01% by weight based on the total weight of the ethanolic extract) (results not shown).

Before animal testing, 1.5 g of ethanol extract was dissolved in 100 ml of solvent (consisting of 95% olive oil and 5% glycerol), and the solution was named "ATG-125" solution.

animal mode - local administration

C57BL/6 male mice (6 and 18 months) were housed in an environment with ad libitum access to food and water, an average constant temperature (26°C) and a light cycle (12 hours light/dark cycle). Mice were randomly divided into four groups (n = 5 per group) containing: (1) Normal control group: aged mice (18 months old) that were continuously fed with water for 30 days (from the 1st day to the 30th day); (2) Sucrose group: Aged mice were continuously fed with 30% sucrose (diluted in water) for 30 days (from day 1 to day 30) to induce sarcopenia, and from day 17 to day 30 locally Administer 100 microliters of olive oil (i.e., 100 microliters of olive oil was administered topically to the abdomen of the mice; as a negative control) once daily; (3) ATG-125 group: aged mice were continuously fed with 30% sucrose (diluted in water) for 30 days (from the 1st day to the 30th day), and were locally administered per kg from the 17th day to the 30th day 60 mg of ATG-125 solution (diluted in olive oil; i.e., 60 mg/kg of ATG-125 solution administered topically to the abdomen of mice) once daily; and (4) 4-OHT group: aged mice were continuously fed with 30% sucrose (diluted in water) for 30 days (from day 1 to day 30), and injected intraperitoneally with 1/kg from day 17 to day 30 mg of 4-hydroxytamoxifen (4-hydroxytamoxifen, 4-OHT; diluted with olive oil; as a positive control), injected once a day.

All mice were sacrificed on day 31. All mice were maintained on a specific diet and treatment until sacrifice. All animal experiments were approved by the Animal Care and Use Committee.

animal mode - oral administration

C57BL/6 male mice (6 and 18 months) were housed in an environment with ad libitum access to food and water, an average constant temperature (26°C) and a light cycle (12 hours light/dark cycle). Mice were randomly divided into four groups (n = 5 per group) containing: (1) Normal control group: aged mice (18 months old) that were continuously fed with water for 30 days (from the 1st day to the 30th day); (2) Sucrose+MPTP group: aged mice were continuously fed with 30% sucrose (diluted in water) for 30 days (from day 1 to day 30), and injected intraperitoneally with 15 per kg from day 11 to day 17 mg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP; dissolved in phosphate buffered saline ), to induce sarcopenia; from the 17th day to the 30th day, 100 microliters of olive oil (as a negative control group) was locally administered to aging mice, once a day; (3) Sucrose+MPTP+ATG-125 group: aged mice were continuously fed with 30% sucrose (diluted with water) for 30 days (from day 1 to day 30), and from day 11 to day 17 in the abdominal cavity Injection of 15 mg/kg MPTP (dissolved in phosphate buffered saline); from day 17 to day 30, aged mice were administered topically 60 mg/kg ATG-125 solution (diluted in olive oil) daily once; and (4) Sucrose+MPTP+Silegerin group: Aged mice were continuously fed with 30% sucrose (diluted with water) for 30 days (from day 1 to day 30), and injected intraperitoneally from day 11 to day 17 15 mg/kg MPTP (dissolved in phosphate buffered saline); from day 17 to day 30 intraperitoneal injection of 1 mg/kg Siligoline (diluted in olive oil) once a day.

All mice were sacrificed on day 31. All mice were maintained on a specific diet and treatment until sacrifice. All animal experiments were approved by the Animal Care and Use Committee.

Western Ink Dot Analysis

Muscle tissue was harvested immediately after sacrificing mice, followed by flash freezing in liquid nitrogen and storage at -80°C. Proteins in muscle tissue were extracted with distilled water containing protease inhibitors, and protein concentrations were determined using a protein assay kit. Proteins (50 μg) were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene difluoride (PVDF) membranes . Immunoblotting was performed with different mouse/rabbit monoclonal or polyclonal antibodies, followed by reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies. According to the user's operating instructions, the reaction is detected by a chemical fluorescence system. The optical intensity of protein bands was quantified by software. Antibodies used in this experiment include: anti-pAMPK antibody, anti-AMPK antibody, anti-pFOXO3a antibody, anti-FOXO3a antibody, anti-MURF1 antibody, anti-β-actin antibody, anti-SIRT1 antibody, anti-PGC1α antibody, anti-UCP1 antibody, anti-UCP2 antibody , anti-UCP3 antibody, anti-pIGF1R antibody, anti-IGF1R antibody, anti-pIRS1 antibody, anti-IRS1 antibody, anti-pPI3K antibody, anti-PI3K antibody, anti-pAKT antibody, anti-AKT antibody, anti-pmTOR antibody, anti-mTOR antibody, anti-pS6K antibody, anti- S6K antibody, anti-p4EBP1 antibody, and anti-4EBP1 antibody.

Histology, Immunohistochemistry and Immunofluorescence Analysis

The muscle tissue of mice was fixed in 4% paraformaldehyde, and after paraffin-embedding, the embedded tissue was cut into tissue sections with a thickness of 5 microns. Before immunostaining, the tissue specimens were deparaffinized with xylene and different concentrations of alcohol according to conventional methods. Use of catalase to block the activity of endogenous peroxidase. Tissue specimens were washed with distilled water and placed in Tris buffer containing 5% normal goat serum (containing 0.5% TWEEN ^® 20, pH 7.4 (TBS-T)) for 30 minutes. After that, the tissue samples were mixed with anti-pAMPK antibody, anti-Glut4 antibody, anti-pFOXO3A antibody, anti-FOXO3A antibody, anti-MuRF1 antibody, anti-PGC1α antibody, anti-UCP1 antibody, anti-UCP2 antibody, anti-UCP3 antibody, anti-pIGF1R antibody, anti-pIRS1 antibody , anti-pPI3K antibody, anti-pAKT antibody, anti-pmTOR antibody, anti-pS6K antibody, anti-p4EBP1 antibody, anti-lipofuscin antibody, and MITOTRACKER ^TM were reacted at room temperature for 2 hours. Add secondary antibody (ALEXAFLUOR ^® 488, ALEXAFLUOR ^® 633, anti-mouse or anti-rabbit antibody) and react at room temperature for 1 hour. All antibodies were diluted with 2% non-immune goat serum in phosphate buffered saline (PBST) containing ^TWEEN® . The tissue samples were reacted with 3,30-diaminobenzidine (DAB) for 5-10 minutes, and then treated with hematoxylin or 4',6-diamidino-2-phenylindole ( 4',6-diamidino-2-phenylindole, DAPI) stained nuclei.

separate RNA and instant PCR analyze

RNA was isolated from 0.2 g of muscle tissue using an RNA extraction kit. For real-time reverse transcription-PCR (qRT-PCR), an equal amount of RNA was reverse transcribed into cDNA using the cDNA reverse transcription kit according to the user's instructions. The levels of atrogin-1 , klotho , TFAM , SIRT1 , ^PGC1α , NRF1 , UCP1 and UCP2 mRNA were analyzed with ^SYBR® Green PCR master mix. After normalization with persistent phenotype genes, the relative expression level was calculated using the ΔΔCt method.

Statistical Analysis

Data are presented as mean ± standard deviation (mean ± SE). The Mann-Whitney rank-sum test and the Wilcoxon rank-sum test were used to compare the data of different groups. P < 0.05 was considered statistically significant.

Example 1 ATG-125 Effects of solution on sarcopenia-related molecules

Sucrose was used to induce sarcopenia in aged mice as described in Materials and Methods. Specific treatments, including olive oil, ATG-125 solution, and 4-OHT, were administered separately to sucrose-induced mice for 14 days. This example will analyze the expression of different molecules associated with sarcopenia, including pAMPK, AMPK, pFOXO3a, FOXO3a, MuRF1, lipofuscin, atrogin-1 and klotho. The results are illustrated in Figures 1 to 3, respectively.

In the muscle tissue of aged mice fed sucrose, adipocytes and degenerated myofibers were observed (Fig. 1). In addition, feeding sucrose increased the protein amounts of pAMPK, FOXO3a, MuRF1, and lipofuscin (four proteins known to cause sarcopenia) in the muscle of mice compared to normal controls (Figures 1 and 2), and mRNA levels of Atrogin-1 and Klotho (the expression of which is known to induce sarcopenia) (Fig. 3). Administration of ATG-125 or 4-OHT treatment reduced adipocyte and degenerated muscle fiber content in muscle and significantly reversed the adverse effects of sucrose on muscle tissue in aging mice (Figures 1-3).

These results confirm that the ATG-125 solution of the present invention can treat sarcopenia by reducing the expression of different molecules in the AMPK, FOXO3a and MuRF1 signaling pathways.

Example 2 ATG-125 solution pair mTOR/ Insulin signaling pathway and its influence on muscle protein synthesis

Impaired insulin signaling (ie, increased blood sugar) is known to cause sarcopenia, and signaling activation of the mTOR/insulin pathway increases muscle protein synthesis, which in turn positively regulates muscle mass in sarcopenic patients. Accordingly, this example will analyze the effects of the ATG-125 solution of the present invention on different molecules in the mTOR/insulin signaling pathway, including IGF1R, IRS1, PI3K, AKT, mTOR, S6K and 4EBP1. The results are illustrated in Figures 4A and 4B, respectively.

As shown in the results in Figures 4A and 4B, the protein levels of pIGF1R, pIRS1, pPI3K, pAKT, pmTOR, pS6 and p4EBP1 (i.e. IGF1R, IRS1, PI3K, AKT, mTOR, S6K and 4EBP1 activation status) was significantly decreased, while administration of ATG-125 or 4-OHT treatment increased the degree of phosphorylation (ie, the degree of activation) of these mTOR/insulin messaging molecules. Unexpectedly, the efficacy of the ATG-125 solution of the present invention was superior to that of 4-OHT (Figures 4A and 4B).

These results confirm that the ATG-125 solution of the present invention can improve the sucrose-induced insulin signaling impairment by up-regulating the mTOR/insulin signaling pathway, thereby increasing the muscle protein synthesis in sucrose-induced mice.

Example 3 ATG-125 Efficacy of solution in improving mitochondrial function

Mitochondria play important roles in energy production, redox balance, and regulation of cell death pathways. It is known that in patients with sarcopenia, mitochondrial morphology and function are significantly altered. Therefore, this example will evaluate whether ATG-125 solution can improve mitochondrial function in animals suffering from sarcopenia. The results are illustrated in Figures 5 and 6, respectively.

Compared to normal controls, sucrose feeding reduced the expression in muscle of various mitochondrial molecules known to regulate mitochondrial biosynthesis and/or function (Figures 5 and 6). Specifically, panels (A) and (B) of Figure 5 indicate that feeding sucrose reduces the mRNA levels of SIRT1 , PGC1α , NRF1 , TFAM , UCP1 , and UCP2 in mouse muscle tissue, respectively. The data in Figure 6 further confirms that sucrose-fed mice had lower levels of SIRT1, PGC1α, UCP1, UCP2, and UCP3 protein expression in muscle tissue compared to water-fed mice. Administration of ATG-125 or 4-OHT treatment increased the expression of these mediators of mitochondrial biosynthesis in muscle tissue of sucrose-induced aging mice (Figures 5 and 6). Notably, administration of ATG-125 resulted in better therapeutic benefit in sucrose-induced mice compared to 4-OHT (Figures 5 and 6).

Example 4 ATG-125 Effects of solution on sarcopenia-related molecules

Sarcopenia was induced in aged mice using sucrose and MPTP as described in Materials and Methods. Specific treatments, including olive oil, ATG-125 solution, and shiligrin, were administered to sucrose- and MPTP-induced mice, respectively, for 14 days. This example will analyze the expression levels of different molecules (including atrogin-1 and klotho) associated with sarcopenia. The results are illustrated in Figure 7.

Degenerated muscle fibers were observed in the muscle tissue of aged mice fed sucrose (Figure 7). In addition, feeding sucrose and MPTP increased the mRNA levels of Atrogin-1 and Klotho in muscle tissue of mice compared to normal controls (the expression of which is known to induce sarcopenia; Figure 7). Treatment with administration of ATG-125 or siligoline reduced the content of degenerated myofibers in muscle and significantly reversed the adverse effects of sucrose and MPTP on muscle tissue in aging mice (Figure 7).

These results demonstrate that the ATG-125 solution of the present invention can treat sarcopenia by downregulating the expression of atrogin-1 and klotho .

Example 5 ATG-125 Efficacy of solution in improving mitochondrial function

Mitochondria play important roles in energy production, redox balance, and regulation of cell death pathways. It is known that in patients with sarcopenia, mitochondrial morphology and function are significantly altered. Therefore, this example will evaluate whether ATG-125 solution can improve mitochondrial function in animals suffering from sarcopenia. The results are illustrated in Figure 8, respectively.

Compared to normal controls, sucrose feeding reduced the expression in muscle of various mitochondrial molecules known to regulate mitochondrial biosynthesis and/or function (Figure 8). Specifically, panels (A) and (B) of Figure 8 indicate that feeding sucrose reduces the mRNA levels of SIRT1 , PGC1α , NRF1 , TFAM , UCP and UCP2 in mouse muscle tissue, respectively. Treatment with administration of ATG-125 or siligoline increased the expression of these mediators of mitochondrial biosynthesis in the muscle tissue of sucrose and MPTP-induced aged mice (Figure 8). Notably, administration of ATG-125 resulted in a better therapeutic benefit in sucrose-induced mice compared to silidrin (Figure 8).

These results indicate that the ATG-125 solution of the present invention can improve mitochondrial function in mice with sucrose and sucrose/MPTP-induced sarcopenia.

Summarizing the above, the present disclosure provides a pharmaceutical composition (ie, ATG-125 solution) that can increase mitochondrial function in sarcopenic animals by modulating the expression of different proteins in sarcopenia pathogenic pathways , to achieve the purpose of treating sarcopenia. Therefore, the pharmaceutical composition of the present invention can be used as a therapeutic agent for preventing and/or treating sarcopenia, thereby improving the life span and quality of life of patients.

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains, without departing from the principle and spirit of the present invention, should Various changes and modifications can be made to it, so the protection scope of the present invention should be defined by the appended claims.

without

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows: FIG. 1 is a representative photograph according to Embodiment 1 of the present disclosure. Regarding the muscle tissue of mice after administration of a specific treatment, among them hematoxylin and eosin (H&E), anti-glucose transporter type 4 (Glut4) antibody, anti-phosphoric acid AMP-activated protein kinase (phosphorylated AMP-activated protein kinase, pAMPK) antibody, anti-phosphorylated forkhead box O3a (phosphorylated forkhead box O3a, pFOXO3a) antibody, anti-FOXO3a antibody, anti-muscle RING-finger Protein-1, MURF1) antibody, or anti-lipofuscin (lipofuscin) antibody to stain muscle tissue; arrows indicate positive cells; 450-490 nm wavelength to detect lipofuscin autofluorescence Scale bar: 100 μm; Figure 2 is based on the results shown in Example 1 of the present disclosure, which illustrates the effect of ATG-125 of the present invention on the protein expression of specific molecules in mouse muscle tissue after administration of specific treatments. Effect; (A) Panel: protein expression of pAMPK, AMPK, pFOXO3a, FOXO3a, MuRF1 and β-actin; (B) Panel: pAMPK on AMPK, pFOXO3a on FOXO3a, and MuRF1 on β-actin The expression ratio was expressed as the mean ± SEM of five independent experiments, and the statistical significance of the mean difference was analyzed using an unpaired Student's t test; * p < 0.05: normal control group versus sucrose group; # p < 0.05: sucrose group vs. sucrose+ATG-125 group; \ P < 0.05: sucrose group vs. sucrose+4-OHT group; Figure 3 is a bar graph according to Example 1 of the present disclosure, It describes the effect of ATG-125 of the present invention on muscle atrophy F box protein ( atrogin-1 ) and klotho mRNA in mouse muscle tissue after administration of specific treatments; the results are expressed as five groups of independent experiments Mean ± SEM, unpaired Student's t test was used to analyze the statistical significance of the mean difference; * p < 0.05: normal control group vs. sucrose group; # p < 0.05: sucrose group vs. sucrose+ATG-125 group; ¥ P <0.05: sucrose group versus sucrose+4-OHT group; Figures 4A and 4B are drawn according to Example 2 of the present disclosure The results are shown, which respectively illustrate the effect of ATG-125 of the present invention on the mTOR/insulin signaling pathway in mouse muscle tissue after administration of specific treatments; Figure 4A: Phosphorylated insulin-like growth factor-1 receptor (phosphorylated insulin-like growth factor-1 receptor) insulin-like growth factor-1 receptor, pIGF1R), IGF1R, phosphorylated insulin receptor substrate 1 (pIRS1), IRS1, phosphorylated phosphoinositide 3-kinase (pPI3K) ), PI3K, phosphorylated AKT (pAKT), AKT, phosphorylated mammalian target of rapamycin (pmTOR), mTOR, phosphorylated ribosomal S6 kinase (pS6K), S6K , phosphorylated eukaryotic translation initiation factor 4E (phosphorylated eukaryotic translation initiation factor 4E, eIF4E) binding protein 1 (p4EBP1), 4EBP1, and the protein expression of β-actin (β-actin); Figure 4B: pIGF1R Expression ratios to IGF1R, and pIRS1 to IRS1 ((A) panels); pPI3K to PI3K, and pAKT to AKT ((B) panels); and pmTOR to mTOR, pS6K to S6K, and p4EBP1 to 4EBP1 Performance ratios ((C) panels); results are presented as the mean ± SEM of five independent experiments, and an unpaired Student's t test was used to analyze the statistical significance of the mean difference; * p < 0.05: normal control group vs. sucrose group; # p < 0.05: sucrose group vs. sucrose+ATG-125 group; ¥ P < 0.05: sucrose group vs. sucrose+4-OHT group; Figure 5 shows the results according to Example 3 of the present disclosure , which illustrates the effect of ATG-125 of the present invention on specific mitochondrial molecules in mouse muscle tissue after administration of specific treatments; (A) small graph: sirtuin 1 ( sirtuin 1, SIRT1 ), increased peroxide levels Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1, PGC1α and cell mRNA content of nuclear respiratory factor 1 (NRF1 ); (B) panel: mitochondrial transcription factor A (TFAM ), uncoupling protein 1 ( UCP1 ) and UCP2 mRNA content; the results were expressed as the mean ± SEM of five independent experiments, and the statistical significance of the mean difference was analyzed by unpaired Student's t test; * p < 0.05: normal control group versus sucrose group; # p < 0.05: sucrose group vs. sucrose+ATG-125 group; \ P < 0.05: sucrose group vs. sucrose+4-OHT group; After specific treatment, the effect of ATG-125 of the present invention on the expression of SIRT1, PGC1α, UCP1, UCP2 and UCP3 protein in mouse muscle tissue; the results are expressed as the mean ± SEM of five independent experiments, using unpaired Student's t test to analyze the statistical significance of the difference in means; * p < 0.05: normal control group vs. sucrose group; # p < 0.05: sucrose group vs. sucrose+ATG-125 group; ¥ P < 0.05: sucrose group vs. sucrose+4 -OHT group; Figure 7 is a representative photograph as described in Example 4 of the present disclosure for muscle tissue of mice after administration of specific treatments; (A) Panel: (H&E) stained muscle Tissue; arrows indicate positive staining cells; scale bar: 100 μm; (B) panel: this histogram illustrates the effect of ATG-125 of the present invention on atrogin-1 and klotho in mouse muscle tissue after administration of a specific treatment The effect of mRNA; results are expressed as the mean ± SEM of five independent experiments, and the statistical significance of the mean difference was analyzed using an unpaired Student's t test; * p < 0.05: normal control group vs. sucrose + MPTP group; # p < 0.05: sucrose+MPTP group vs. sucrose+MPTP+ATG-125 group; ¥ P <0.05: sucrose group vs. sucrose+MPTP+selegiline group; and FIG. 8 is according to Example 5 of the present disclosure. The results shown, which illustrate the effects of ATG-125 of the present invention on specific mitochondrial molecules in mouse muscle tissue after administration of specific treatments; (A) Panel: mRNA content of SIRT1 , PGC1α and NRF1 ; (B) ) panels: mRNA levels of TFAM , UCP1 and UCP2 ; results are presented as the mean ± SEM of five independent experiments, and the unpaired Student's t test was used to analyze all Statistical significance of difference in value; * p < 0.05: normal control group vs. sucrose+MPTP group; # p < 0.05: sucrose+MPTP group vs. sucrose+MPTP+ATG-125 group; ¥ P < 0.05: sucrose group vs. Sucrose + MPTP + Hilledrin group.

Claims (15)

A pharmaceutical composition for treating sarcopenia, comprising: Chlorogenic acid, leonurine, schaftoside, rutin, schaftoside, chlorogenic acid, 4,5 - 4,5-dicaffeoylquinic acid, quercetin, apigenin, glycyrrhizic acid, bisdemethoxycurcumin, demethoxycurcumin mixtures of demethoxycurcumin, curcumin and artemisetin; and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 1, wherein the mixture comprises 5-10 weight percent of the chlorogenic acid, 0.1-2 weight percent of the leonurine, 0.1-2 weight percent of the saftoside, 5- 10 weight percent of the rutin, 35-45 weight percent of the isoxafutoside, 20-30 weight percent of the isochlorogenic acid, 3-6 weight percent of the 4,5-dicaffeoquinic acid, The quercetin of 0.1-0.5 weight percent, the apigenin of 1-3 weight percent, the glycyrrhizin of 1-3 weight percent, the two-demethoxycurcumin of 1-3 weight percent, 1-3 Weight percent of the demethoxycurcumin, 5-10 weight percent of the curcumin, and 0.1-0.5 percent by weight of the eflavin. The pharmaceutical composition of claim 2, wherein the mixture comprises 7-8 weight percent of the chlorogenic acid, 0.5-1 weight percent of the leonurine, 0.5-1.5 weight percent of the saftoside, 7- 8 weight percent of the rutin, 38-42 weight percent of the isosafutoside, 20-25 weight percent of the isochlorogenic acid, 4-5 weight percent of the 4,5-dicaffeoquinic acid, The quercetin of 0.1-0.3 weight percent, the apigenin of 1-2 weight percent, the glycyrrhizin of 1-2 weight percent, the two-demethoxycurcumin of 2-3 weight percent, 2-3 Weight percent of the demethoxycurcumin, 5-7 weight percent of the curcumin, and 0.1-0.3 weight percent of the eflavin. The pharmaceutical composition of claim 1, wherein the mixture is at a temperature of about 30-100° C., using ethanol to extract mugwort ( Artemisia argyi ), mulberry ( Morus alba L. ), motherwort ( Leonurus japonicus Houtt ) , pepper ( Capsicum annuum L. ), bamboo leaves ( Lophatherum gracile Brongn ), turmeric ( Curcuma longa ) and licorice ( Glycyrrhiza uralensis ) for 0.5-5 hours. The pharmaceutical composition as claimed in claim 4, wherein the mixture is at a temperature of about 50-80° C., using 95% ethanol to extract the leaves of the mugwort, the leaves of the mulberry, the leaves of the motherwort, the bell pepper The leaves of the bamboo, the leaves of the pale bamboo leaves, the roots of the Sichuan turmeric and the roots of the licorice are prepared for 3-5 hours. The pharmaceutical composition of claim 5, wherein in the extraction process, the leaves of the mugwort, the leaves of the mulberry, the leaves of the motherwort, the leaves of the pepper, the leaves of the pale bamboo, the roots of the turmeric And the root of the licorice is mixed in a weight ratio of about 4-6:4-6:4-6:2-3:2-3:1:1. The pharmaceutical composition of claim 6, wherein the leaves of the mugwort, the leaves of the mulberry, the leaves of the motherwort, the leaves of the pepper, the leaves of the pale bamboo, the roots of the turmeric and the roots of the licorice It is mixed in a weight ratio of about 5:5:5:2.5:2.5:1:1. A pharmaceutical composition for treating sarcopenia, comprising: An ethanolic extract of a Chinese herbal medicine mixture, wherein the Chinese herbal medicine mixture is composed of mugwort, mulberry, motherwort, bell pepper, pale bamboo leaf, sichuan turmeric and licorice; and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 8, wherein the Chinese herbal medicine mixture is composed of the leaves of the mugwort, the leaves of the mulberry, the leaves of the motherwort, the leaves of the pepper, the leaves of the pale bamboo, the roots of the turmeric and the root of the licorice. The pharmaceutical composition of claim 9, wherein in the Chinese herbal medicine mixture, the leaves of the mugwort, the leaves of the mulberry, the leaves of the motherwort, the leaves of the pepper, the leaves of the pale bamboo, the leaves of the turmeric The weight ratio of the root to the root of the licorice is about 4-6:4-6:4-6:2-3:2-3:1:1. The pharmaceutical composition of claim 10, wherein the weight ratio is about 5:5:5:2.5:2.5:1:1. Use of the pharmaceutical composition according to claim 1 or claim 8 for preparing a medicament for treating sarcopenia in an individual. The use of claim 12, wherein the medicament is administered orally or topically to the individual. The use of claim 13, wherein the medicament is administered to the individual daily for at least 7 days. The use of claim 14, wherein the medicament is administered to the individual daily for at least 14 days.

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